The Turnbull Government's same-sex marriage plebiscite bill has failed to pass the Senate in a late night vote.
The bill was defeated at the second reading on Monday night, with 33 votes against the bill to 29 in support.
It was looking unlikely to pass after the Nick Xenophon Team confirmed they would vote against the bill earlier on Monday.
On Monday night, the federal government had the support of David Leyonhjelm and Jacqui Lambie and One Nation senators. Senators voting down the plebiscite bill included Labor, the Greens and the Nick Xenaphon Team.
The bill, which would have had the Australian public vote on marriage equality in February, passed the House of Representatives in October but has been deemed doomed after Labor pledged to vote against it weeks ago.
The plebiscite has been highly debated for months, with the LGBTIQ community deeming it destructive and potentially provoking hate speech.
Critics also criticised the move as an expensive poll, as the plebiscite would have cost around $200 million and does not guarantee legislative change as, unlike a referendum, it is not legally binding.
Prime Minister Malcolm Turnbull has warned that marriage equality could be delayed for years if the plebiscite does not pass.
The Greens have already labelled the defeat a win for equality and justice with the Greens LGBTIQ spokeswoman Senator Janet Rice claiming "tonight we moved forward to equality".
"The Greens are proud to have stood with lesbian, gay, bisexual, trans, intersex and queer communities against this plebiscite and we recognise, share and draw strength from the determination of this community to achieve the equality everyone deserves," Rice said in a statement.
The defeat was another blow for the federal government after proposed media reforms were also defeated in a late night vote, after Labor opposed the bill.
Communications Minister Mitch Fifield slammed labor for "supporting analogue laws in a digital world" in a statement on Monday night.
Currently the "two out of three" rule exists which confirms one person owning a television station, radio station and newspaper in a single city.